The goal of the work proposed in this application is to continue studies aimed defining the amino acid residues involved in the catalytic mechanism of prostaglandin endoperoxidase G/H synthase. PGG/H synthase catalyzes the first step in the conversion of arachidonic acid to prostacyclin and thromboxane A2, two agents which play important roles in hemostasis. PGG/H synthase is a hemoprotein which has two activities, including a cyclooxygenase and a hydroperoxidase activity. In work performed previously, we have deduced the amino acid sequences of the sheep and mouse PGG/H synthases and a portion of the human PGG/H synthase. Based on sequence comparisons among PGG/H synthases and other peroxidases, we have proposed that His226 and His3O9 are the distal and proximal heme ligands of PGG/H synthase. We have established that the "active site" serine which is acetylated by aspirin is Ser530, and using site-directed mutagenesis, we have provided evidence that acetylation of Ser530 places a bulky group at the active site of the cyclooxygenase, thereby interfering with arachidonate binding. Using modification with tetranitromethane, we have also recently identified what is likely to be a tyrosine at the active site of the cyclooxygenase. We now propose: (a) to use site-directed mutagenesis to determine if His3O9 and His226 are specifically involved in heme binding to PGG/H synthase, and further, whether residues adjoining these histidines are important in heme binding. (b) to use site-directed mutagenesis to determine if the amino acids adjoining Ser530 play important roles in arachidonate binding. (c) to identify the tyrosine residue which is modified by tetranitromethane to cause inactivation of the cyclooxygenase.